Together with signaling through the T cell antigen receptor, costimulation is essential for a robust and properly regulated immune response. The engagement of CD28 and ICOS with the B7 isoforms and B7H, respectively, on antigen presenting cells, provide the stimulatory signals that direct T cell proliferation and cytokine production. Engagement of CTLA-4 and PD-1 with the B7 and PD-L isoforms, respectively, on antigen presenting cells, provides inhibitory signals required for attenuation of the T cell response and the induction of peripheral tolerance. As a consequence of their fundamental roles in modulating T cell responsiveness, these costimulatory molecules and their associated signaling pathways are active targets for the development of therapeutics to treat a wide range of human pathologies, including cancer, autoimmunity, and graft rejection. Our recent structural studies have identified a highly ordered, alternating network of CTLA-4 and B7-2 homodimers that for the first time provides a model for the organization of these molecules and their associated signaling partners within the T cell/antigen presenting cell immunological synapse. These observations have generated a number of directly testable hypotheses regarding the molecular mechanisms of CTLA-4 costimulation. Our overall goals are to understand the features of the CTLA-4 and B7 homodimers that contribute to signaling, the importance of the periodic organization of the CTLA-4/B7 assembly for function, and the generality of these features in the signaling mechanisms of the other costimulatory receptor-ligand pairs. We have adopted a multidisciplinary approach that combines three-dimensional structural, biochemical, and cell biological information with appropriate mammalian models so as to determine in vivo structure-function correlations for the CD28/CTLA-4/ICOS family of costimulatory molecules. Accordingly, our Specific Aims are: 1) Three dimensional structure determination of the costimulatory receptor-ligand pairs; 2) Characterization of the biologically relevant oligomeric states of the costimulatory molecules; 3) Identification of the functional requirements associated with dimeric organization that are relevant to costimulation; 4) Examination of the organization of costimulatory molecules at the immunological synapse.